Eukaryotic RNA is synthesized with a 5′ cap consisting of a guanine nucleotide linked via a 5′ to 5′ triphosphate linkage to the penultimate nucleotide. In prokaryote, the 5′ terminus of a messenger RNA (mRNA) consists of a triphosphate group, without the guanine nucleotide.
The process of 5′ capping is required for creating mature mRNA which is then able to undergo translation. Capping enhances RNA stability. In vitro manipulation of the RNA often requires removal of the 5′ guanine cap.
Hydrolysis of the triphosphate by chemical means is straight forward, but is not readily controlled and can result in digestion of the entire RNA. Enzymes have been described for removing the 5′ cap by selectively hydrolyzing the 5′ to 5′ triphosphate linkage leaving the remaining RNA intact. Tobacco acid pyrophosphatase (TAP) is the most widely used enzyme at present (Lockard et al., Gene Amplification and Analysis, 2:229-251, (1981)). TAP has not been cloned and is difficult to prepare. Other enzymes capable of removing a 5′ cap and each having certain limitations include D10 (Souliere, et al., Biochemical Journal, 420:27-35, (2009)), Dcp1p (LaGrandeur and Parker, The EMBO Journal 17:1487-1496, (1998), Liu et al., The EMBO Journal, 21:4699-4708, (2002)), hNUDT16(Lu, et al., Protein Cell, 2:64-73, (2011)), Nhm1 (Salehi, et al., Molecular Microbiology, 46:49-62, (2002)) and Rai 1 (Jiao, et al., Nature, 467:608-611, (2010)). Except for Rai 1, these enzymes are active only for 5′ cap that consists of a 7-methylated guanine nucleotide and not for an unmethylated guanine cap. Moreover, most of these enzymes are difficult to clone. It would be desirable to identify a decapping enzyme that can be easily produced in large quantities, and can remove the guanine cap from an RNA whether it is methylated or unmethylated.